This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Prevention of chronic kidney disease (CKD) is a major public health issue. Although
several studies have been performed on the association between alcohol consumption
and CKD or renal function, it remains controversial. Numerous genetic polymorphisms
have been reported to be associated with CKD and kidney function. Mitochondrial DNA
cytosine/adenine (Mt5178 C/A) polymorphism is associated with longevity in Japanese.
This polymorphism modifies the effects of alcohol consumption on blood pressure, risk
of hypertension, serum triglyceride levels, risk of hyper-LDL cholesterolemia and
serum uric acid levels. The objective of this study was to investigate whether Mt5178
C/A polymorphism modifies the effects of alcohol consumption on renal function in
male Japanese health check-up examinees.

Methods

A total of 394 male subjects aged 29–76 years were selected from among individuals
visiting the hospital for regular medical check-ups. After Mt5178 C/A genotyping,
a cross-sectional study assessing the combined effects of Mt5178 C/A polymorphism
and habitual drinking on the risk of mildly decreased estimated glomerular filtration
rate (eGFR) (<90 ml/min/1.73 m2) was conducted.

Keywords:

Background

Prevention of chronic kidney disease (CKD) is a major public health issue. Similarly
to the survey reporting the prevalence of CKD in the United States [1], analysis of data, sampled from over half a million individuals in the general Japanese
population, predicted that about 13% of the Japanese adult population had CKD in 2005
[2]. Considering that major outcomes of CKD are loss of kidney function and cardiovascular
disease [3], primary prevention for CKD is required. Therefore, it is crucial to detect the modifiable
risk factors of CKD using epidemiological approaches.

There have been several epidemiological studies on the association between alcohol
consumption and CKD. A case–control study did not show an increased risk of CKD associated
with alcohol consumption [4]. Another case–control study reported that consumption of more than two alcoholic
drinks per day was associated with the risk of end-stage renal disease [5]. Both cross-sectional and longitudinal analyses in a population-based cohort showed
an independent association between heavy alcohol consumption and CKD [6]. However, a large prospective cohort study suggested an inverse relationship between
moderate alcohol consumption and the risk of renal dysfunction [7]. A large-scale cross-sectional study also showed an inverse association between frequency
of alcohol consumption and CKD in healthy Japanese men [8]. These epidemiological studies did not assess genetic factors.

Mitochondrial DNA cytosine/adenine (Mt5178 C/A) polymorphism, which is also recognized
as NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism,
is associated with longevity in Japanese [9]. The frequency of the Mt5178A genotype is significantly higher in Japanese centenarians
than in the general population. Moreover, Japanese individuals with Mt5178A are more
resistant to adult-onset diseases, such as hypertension [10], diabetes [11], myocardial infarction [12,13] and cerebrovascular disorders [14], than those with Mt5178C. This polymorphism modifies the effects of alcohol consumption
on blood pressure [15], risk of hypertension [10], serum triglyceride levels [16], risk of hyper-LDL cholesterolemia [17] and serum uric acid levels [18]. In addition to these previous reports [10,15-18], several molecular epidemiological studies on CKD and kidney function [19-25] have encouraged us to examine the joint effects of Mt5178 C/A polymorphism and alcohol
consumption on renal function.

In this study, we investigated whether longevity-associated Mt5178 C/A polymorphism
modifies the effects of habitual alcohol consumption on renal function in male Japanese
health check-up examinees.

Methods

Subjects

Participants were recruited from among individuals visiting the Mito Red Cross Hospital
for regular medical check-ups between August 1999 and August 2000. This study was
conducted in accordance with the Declaration of Helsinki and was approved by the Ethics
Committee of the Kyorin University School of Medicine. Written informed consent was
obtained from 602 volunteers before participation. Because the number of women was
insufficient for classification into groups based on Mt5178 C/A genotype and alcohol
consumption, women were excluded. Due to glomerular hyperfiltration in early diabetes
[26], diabetic patients undergoing treatment were also excluded. Thus, 406 men were enrolled
in the study. Twelve individuals with unclear data were subsequently excluded; therefore,
subjects comprised 394 Japanese men aged 29–76 years.

Clinical characteristics of subjects

Data on age, height, weight, blood pressure, serum lipid level, fasting plasma glucose
level, serum uric acid level, blood urea nitrogen level and serum creatinine level
were collected from the results of regular medical check-ups. Renal function was evaluated
by estimated glomerular filtration rate (eGFR), which was calculated using a three-variable
Japanese equation: eGFR = 194 × creatinine− 1.094 × age− 0.287[27]. Similarly to other genetic epidemiological studies [19,20], based on K/DOQI CKD classification [3], reduced eGFR was considered to be <90 ml/min/1.73 m2. Body mass index (BMI) was defined as the ratio of subject weight (kg) to the square
of subject height (m). A survey of alcohol consumption, habitual smoking, coffee consumption,
medical history and medication use was performed by means of questionnaire. Alcohol
consumption was classified based on drinking frequency (daily drinkers; occasional
drinkers, which include those who drink several times per week or per month; and non-
or ex-drinkers). Smoking status was classified based on number of cigarettes smoked
per day (never- or ex-smokers; 1–20 cigarettes smoked per day; and >20 cigarettes
smoked per day). Coffee consumption was classified based on number of cups of coffee
per day (≤1 cup per day; 2–3 cups per day; and ≥4 cups per day). For use of antihypertensive
medication, subjects were classified as taking no drug treatment or taking medicine.

Genotyping

DNA was extracted from white blood cells using the DNA Extractor WB kit (Wako Pure
Chemical Industries, Osaka, Japan). The Mt5178 C/A polymorphism was detected by polymerase
chain reaction (PCR) and digestion with AluI restriction enzyme. The sequence of primers was: forward 5′-CTTAGCATACTCCTCAATTACCC-3′, reverse 5′-GTGAATTCTTCGATAATGGCCCA-3′. PCR was performed with 50 ng of genomic DNA in buffer containing each primer at
0.2 μmol/l, 1.25 mmol/l dNTPs, 1.5 mmol/l MgCl2, and 1 U of Taq DNA polymerase. After initial denaturation at 94°C for 5 min, PCR
was conducted through 40 cycles as follows: denaturation at 94°C for 30 s, annealing
at 60°C for 60 s and polymerase extension at 72°C for 90 s. After cycling, a final
extension at 72°C for 10 min was performed. PCR products were digested with AluI restriction enzyme (Nippon Gene, Tokyo, Japan) at 37°C overnight, and were electrophoresed
on 1.5% agarose gels stained with ethidium bromide for visualization under ultraviolet
light. The absence of an AluI site was designated as Mt5178A, and the presence of this restriction site was designated
as Mt5178C.

Statistical analyses

Statistical analyses were performed using SAS statistical software, version 9.2 for
Windows. Multiple logistic regression analysis was used to calculate odds ratios (OR)
for the reduction of eGFR (<90 ml/min/1.73 m2). Covariates were selected for their ability to confound the associations as determined
through univariate and stepwise models. For multiple logistic regression analysis
and analysis of covariance, habitual smoking (never- or ex-smokers = 0; 1–20 cigarettes
smoked per day = 1; >20 cigarettes smoked per day = 2), coffee consumption (≤1 cup
per day = 1; 2–3 cups per day =2; ≥4 cups per day = 3) and antihypertensive medication
use (no use of antihypertensive = 0; use of antihypertensive = 1) were numerically
coded. Differences with P values of less than 0.05 were considered to be statistically
significant.

Results

No significant differences in age, BMI, systolic blood pressure, diastolic blood pressure,
serum total cholesterol levels, serum HDL cholesterol levels, serum triglyceride levels,
fasting plasma glucose levels, serum uric acid levels or blood urea nitrogen levels
were observed between the Mt5178C and Mt5178A genotypes (Table 1). There were no outliers in eGFR for either genotype; eGFR in Mt5178C genotypic men
ranged from 50 to 119 ml/min/1.73 m2 and that in Mt5178A genotypic men ranged from 52 to 112 ml/min/1.73 m2. Among men with Mt5178C, the frequency of daily drinkers was 46.4%, that of occasional
drinkers was 35.2% and that of non-drinkers was 18.4%. Among those with Mt5178A, the
frequency of daily drinkers was 47.7%, that of occasional drinkers was 38.7% and that
of non-drinkers was 13.6%. Chi-squared test showed no significant differences in alcohol
consumption between the Mt5178 C/A genotypes (P = 0.426).

For Mt5178A genotypic men, the frequency of alcohol consumption was positively and
significantly associated with eGFR (P for trend = 0.003) (Table 2). After adjustment, this positive association between alcohol consumption and eGFR
remained significant. Moreover, eGFR was significantly higher in daily drinkers than
in non-drinkers (P = 0.005). After adjustment for age and BMI or for age, BMI, habitual smoking, coffee
consumption and use of antihypertensive medication, eGFR was also significantly higher
in daily drinkers than in non-drinkers (P = 0.025 and P = 0.026, respectively). On the other hand, although eGFR was significantly higher
in occasional drinkers with Mt5178C than in non-drinkers with Mt5178C (P = 0.043), the association between Mt5178C genotype and eGFR did not appear to depend
on the frequency of alcohol intake.

For subjects with Mt5178A, the risk of decreased eGFR may depend on frequency of alcohol
consumption (P for trend = 0.003) (Table 3). After adjustment, the negative association between increasing frequency of alcohol
consumption and the risk of decreased eGFR remained significant. The crude OR for
decreased eGFR was significantly lower in daily drinkers than in non-drinkers (OR
= 0.092, 95% confidence interval (CI): 0.012–0.727, P = 0.024). After adjustment for age and BMI or for age, BMI, habitual alcohol consumption,
coffee consumption and use of antihypertensive medication, a significant OR remained
(adjusted OR = 0.098, 95% CI: 0.012–0.784, P = 0.029 and adjusted OR = 0.091, 95% CI: 0.011–0.747, P = 0.026, respectively). On the other hand, the association between Mt5178C genotype
and the risk of decreased eGFR does not appear to depend on alcohol consumption.

Discussion

In the present study, we observed that Mt5178 C/A polymorphism apparently modifies
the effects of habitual alcohol drinking on eGFR in Japanese men. This is a new gene-environment
interaction on renal function. For men with Mt5178A, habitual alcohol consumption
may reduce the risk of mildly decreased eGFR. On the other hand, for those with Mt5178C,
alcohol consumption does not appear to influence eGFR.

Schaeffner et al. reported an inverse association between moderate alcohol consumption
and the subsequent risk of renal dysfunction in large cohort of apparently healthy
men [7]. They used eGFR calculated by the Cockcroft-Gault equation, and reported that men
who consumed at least seven drinks per week had an approximately 25% lower risk of
reduced eGFR (<55 ml/min) in a 14-year period than those who consumed one or fewer
drinks per week. Funakoshi et al. also revealed an inverse relationship between frequency
of alcohol consumption and CKD in apparently healthy men [8]. They used eGFR calculated using the three-variable Japanese equation and found that
everyday drinkers had an approximately 40% lower risk of CKD (eGFR <60 ml/min/1.73 m2) than non-drinkers. Judging from both investigations [7,8], alcohol consumption was observed to have a desirable effect on the risk of decreased
eGFR. However, our observations suggest that genetic information is required to assess
the relationship between alcohol intake and renal function.

From the viewpoint of preventing mildly decreased eGFR, habitual drinking appears
to be beneficial for Mt5178A genotypic men. Moreover, for men with Mt5178A, alcohol
consumption decreases serum triglyceride levels [16]. However, it is uncertain whether alcohol consumption is beneficial overall for Mt5178A
genotypic men. In contrast, for Mt5178C genotypic men, daily alcohol consumption may
increase the risks of hypertension [10] and hyperuricemia [18], but may also reduce the risk of hyper-LDL cholesterolemia [17]. Thus, it is unclear whether alcohol consumption is detrimental for Mt5178C genotypic
men. Although further genetic epidemiological research is necessary, genotyping of
Mt5178 C/A is thought to be practical for personalized prevention of lifestyle-related
diseases, including CKD.

The mechanisms underlying the joint effects of Mt5178 C/A polymorphism and alcohol
consumption on renal function have not been elucidated. This gene-environment interaction
is presumed to result from differences in the biophysical and biochemical properties
of ND2-237 Leu/Met. NADH dehydrogenase is regarded as the major physiological and
pathological site of reactive oxygen species (ROS) generation in mitochondria, and
as a target of assault by ROS [28]. Mouse mitochondrial DNA 4738 C/A (Mt4738 C/A) polymorphism leads to a leucine to
methionine substitution in NADH dehydrogenase subunit 2. ROS production by NADH dehydrogenase
is significantly lower in mice with Mt4738A than in those with Mt4738C [29]. The results in experimental mice indicate that ND2-237Met suppresses ROS production
in humans. Moreover, as methionine residues play an antioxidant role in scavenging
ROS [30], ND2-237Met may also protect NADH dehydrogenase itself from ROS attack.

Ethanol metabolism is directly involved in the production of ROS [31]. There may be biophysical and biochemical differences in the protection against ethanol-induced
ROS or the reduction of ethanol-induced ROS generation between ND2-237Leu and ND2-237Met.
These apparent disparities are thought to result in the combined effects of Mt5178
C/A polymorphism and habitual drinking on eGFR. Recently, using male C57BL/6 mice,
Yuan et al. demonstrated that moderate ethanol exposure can provide protection for
kidneys against ischemia/reperfusion-induced renal injury by enhancing antioxidant
capacity characterized by higher activity of superoxide dismutase, which is a critical
enzyme responsible for detoxifying ROS [32]. However, to determine the mechanisms responsible for interaction between ND2-237
Leu/Met genotypes and alcohol consumption on renal function, further biochemical and
pharmacological studies are necessary.

In the Japanese population, several genetic polymorphisms have been reported to be
associated with CKD, defined as eGFR <50 ml/min/1.73 m2[21,22] or <60 ml/min/1.73 m2[23,24]. In addition, promoter polymorphism of the endotoxin receptor [19], single nucleotide polymorphism rs1287637 in the nephronophthisis 4 gene is associated
with mildly decreased eGFR (<90 ml/min/1.73 m2) [20]. Therefore, these molecular epidemiological studies, as well as our results, indicate
gene-gene or gene-gene-environment interactions on renal function.

In accordance with K/DOQI CKD classification [3], eGFR of <90 ml/min/1.73 m2 was defined as reduced eGFR in this cross-sectional study. In the present subjects,
the prevalence of eGFR of <90 ml/min/1.73 m2 was 82.4% in men with Mt5178C and was 74.8% in those with Mt5178A. A large-scale
population-based epidemiological study showed that the prevalence of eGFR of <90 ml/min/1.73 m2 was 80.6% in Japanese men aged 30–69 years [2]. Therefore, whether the validity of the definition of reduced eGFR adopted in our
study is worthy of further consideration.

There are several important limitations in this study. First, as compared with other
genetic epidemiological studies on renal function [19-25], the study sample was too small. Second, selection bias is likely due to the recruiting
of subjects from those visiting the hospital for regular medical check-ups, and the
prevalence of moderately decreased eGFR of <60 ml/min/1.73 m2, recognized as CKD, was low among the study subjects. Third, this study was a cross-sectional
study, and although the study design can suggest causal links, it cannot establish
valid causality. To overcome these limitations, a large-scale population-based follow-up
study is necessary. Fourth, because of lack of data on the amount of alcohol intake,
the evaluation of habitual alcohol intake was based on the frequency of alcohol consumption.
Although we have also used this evaluation method in previous studies [10,15-18], the existence of joint effects between Mt5178 C/A polymorphism and volume of alcohol
intake on eGFR warrants further investigation. Finally, we lacked information on proteinuria,
which is an early and sensitive marker of kidney damage in various types of CKD [3].

Conclusion

This study identified a novel joint effect by a genetic factor and lifestyle behavior
on renal function. Longevity-associated Mt5178 C/A polymorphism may modulate the effects
of alcohol consumption on eGFR or the risk of mildly decreased eGFR in Japanese men.
For Mt5178A genotypic men, habitual drinking may increase eGFR or reduce the risk
of mildly decreased eGFR. On the other hand, for Mt5178C genotypic men, habitual drinking
does not appear to affect eGFR. Together with a complete evaluation of the biophysical
or biochemical effects of alcohol intake, genetic information on Mt5178 C/A polymorphism
may contribute to primary individualized prevention of CKD and consequential reductions
in either kidney dysfunction or cardiovascular disease.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AK designed the study, carried out the epidemiological survey, carried out the genotyping,
analyzed the data, and drafted the manuscript; MI collected the samples; NM assisted
with genotyping; KK and MY carried out the epidemiological survey; NS, TO, TS, HO
and HH assisted in data analysis and interpretation; YT designed the study and carried
out the epidemiological survey. All authors have read and approved the final manuscript.

Acknowledgements

This study was supported in part by Grants-in-Aid from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (No. 14570355, No. 18590572 and No.
23500859) and the Chiyoda Mutual Life Foundation.